Over 40 million bases of genetic code originating from various organisms from humans to virus have been elucidated in the laboratory over the last decade. One of the objectives of the Human Genome Project is to sequence the approximately 3 billion bases that make up the human genome. Obviously, significant advances need to be made in DNA sequencing technology if this goal is ever to be achieved in a reasonable time.
The standard electrophoresis method is separation through a continuous polyacrylamide slab gel. In the preparation of a gel slab the liquid polyacrylamide gel is poured as a single continuous sheet between two glass plates separated by spacers at the boundaries. When the gel polymerizes a glass plate-gel-glass plate sandwich is formed. Sample loading slots are cast in the gel using a plastic comb shaped device set in place before the gel polymerizes and removed before loading the samples. This format is limited in the degree of data compaction that can be achieved in one run due to difficulties in lateral stability (sample drift) that occurs if the sample wells are placed any closer than 0.2 mm to one another and are any smaller than 4.0 mm in width. As a consequence of this limitation, improvements in slab technology have centered around decreases in running time and increases in the degree of resolution rather than increases in data compaction. The most significant advancement in this direction is the development of ultra-thin slab gels using extremely thin spacers that increase resolution and decrease running times.
A technology that does not appear to be limited by data compaction considerations is the recent development of gel-filled capillaries. Each capillary is an isolated electrophoresis unit that accepts a single sample and is limited theoretically only by the dimensions of the capillary. The large surface to volume ratio of the capillary allows highly efficient dissipation of electro-resistant generated heat. The greater the de-coupling of gel temperature from electro-resistive generated energy the greater the effective field strength that can be applied. The linear range over which migration rate varies directly with field strength is extended using gel filled capillaries allowing separations at higher fields with greatly reduced running times.
The advantage in a single capillary, however, must somehow be translated to a large array of coupled capillaries each with the same characteristics which can be electrophoresed together under identical conditions to make this technology viable for DNA sequencing and to achieve significant data compaction. The creation of such an ensemble is a complex technical challenge that limits the usefulness of gel filled capillaries in real sequencing applications.
The ribbon channel plate of the instant invention has both the data compaction and energy dissipation characteristics of gel filled capillaries and the ease of preparation, reliability, and easy recasting characteristics of slab gels.
Current DNA sequencing chemistries establish the sequence of nucleotides (bases) along a DNA molecule by subjecting four sets of identical molecules that have been referenced chemically from the same point on the same end to a series of reactions. In one tube is an A (Adenine) specific, in one a T (Thymidine) specific, in one a G (Guanine) specific and in one a C (Cytosine) specific reaction. In each tube a set of molecular fragments is formed with each nucleotide position as terminator to a portion of the population. These reaction mixtures are then simultaneously electrophoresed through a gel where the populations separate into distinct bands of identical size fragments migrating inversely to their size (molecular weight) with the smaller fragments bands moving at faster rates than the larger ones. The set of resolved bands is called the sequence ladder. In the standard procedure,each of four sample wells formed in the gel when it sets up, is loaded with one of the nucleotide specific reaction mixtures. Upon simultaneous electrophoresis bands will form of size determined by the dimensions of the gel sample wells and will migrate in the order of the DNA sequence.
These fragment bands that make up the ladders can be labeled before separation with either radioactive or florescence labels. Another technique is to label each of the four types of nucleotide termination with a specific florescent molecule. Since the emission spectra of each label is different, a mixture of all four reactions can be electrophoresed from the same well in the gel effectively increasing data compaction four fold. In other techniques the resolved bands can be elucidated by hybridizing electro-eluted fragments on deposition membranes to specific labeled DNA fragment probes.
Labeled DNA probes can be hybridized to the sequence ladder, washed off, and then the ladder probed again. This recursive probe hybridization procedure is the basis for the Church multiplexing technique in which the data compaction can be increased up to 40 fold. In multiplexing, up to 40 different DNA samples of different sequence are mixed together prior to performing the DNA sequencing reactions. These samples are then processed together as if they were one. At the end of the procedure, the individual ladders associated with a particular sequence are revealed by hybridization with a labeled DNA probe specific for that sequence. The probe is then washed off and a new probe is applied to reveal a different sequence. The process is repeated until all 40 starting sequences are revealed.
The transfer of DNA fragments to nylon or other suitable support materials, such as nitrocellulose, is an essential component of all DNA hybridization techniques including in addition to multiplex sequencing, chemiluminescence detection, DNA finger printing, and various other techniques that are not necessarily DNA sequencing procedures. The DNA fragments are transferred to deposition membranes either by various elution techniques or by electrophoresing the bands completely off the gel onto a moving deposition membrane, a process called direct blotting.
Conventional direct blotting devices utilize a deposition membrane attached to a conveyor belt in the lower buffer chamber that remains totally submerged during electrophoresis. Such devices do not allow detection or downstream processing except by physical removal of the nylon or other membrane from the conveyor belt. The almost total submersion of the deposition membrane makes detection and processing in the electrophoretic device proper a difficult if not impossible task that must take place through the liquid buffer.
A component of the instant invention is a unique drum electrophoresis unit which allows direct blotting of the sequence ladders onto a nylon or other suitable deposit membrane affixed to a rotating drum. The deposition interface between the end of the plate supporting the gel and the membrane covered drum is not submerged in buffer but is removed to a distance away from the lower buffer chamber. This invention conveys significant advantages to the system as compared with submersed interface and conveyor belt direct blotting systems.
In the instant invention the drum is only partially immersed in the bottom buffer chamber and can easily be removed without disturbing the deposition membrane. The drum can then be moved intact to suitable downstream detection and processing units which can be rugged and reliable because of the enabling geometry of the deposition membrane attached to the drum.
The deposited DNA ladder in the instant invention, spends considerable time in the open non-buffer space outside of the lower buffer chamber. Because of lack of buffer in this space processing devices such as UV fixing lamps or radiation or florescence detection devices can be built into the electrophoresis device that allow detection and part or all of the downstream processing to occur in a single device without moving the drum. Alternatively, the drum can be easily removed with attached membrane and easily moved to specially designed downstream detection and processing units.
Productivity is currently limited by the amount of material that can be processed at one time--that is, during a single run. The lower limit for width of a single slab-gel loading well on a conventional DNA sequencing apparatus is approximately 4 mm, with a required spacing between adjacent wells of about 0.2 mm. If these dimensions are decreased to increase data compaction, significant ladder drift and data cross talk occur during electrophoresis which severely limits the accuracy of the data. With these limitations only about 48 lanes can be run on a single nylon membrane with about 200 mm of effective deposition surface affixed to the drum of the instant invention.
Another aspect of the instant invention is a component called a ribbon channel plate which provides increased productivity in terms of data density packed into a single run with concomitant increases in sequencing speed and data resolution. The ribbon channel plate consists of a plate with a series of adjacent micro channels which can replace the conventional slab gel. When cast with gel, each micro-channel represents an electrophoresis lane isolated and independent of its neighbors which eliminates the common problem of drift and interference that limits data compaction of standard continuous slab gels when loading density is increased.
Other aspects of the device enables efficient processing in the identification of DNA sequences by any one of the available detection methods including multiplex sequencing. Still another aspect is an increase of speed in DNA sequencing because of the ability to run at higher electric fields with efficient temperature control of the ribbon channel plate.
The human genome consists of three billion bases distributed over 24 chromosomes. The invention herein described, under optimal conditions can process about 24,000 (48 reaction sets (with each reaction set occupying 4 lanes), X 500 bases per sequence length) bases per run. The adaptation to multiplexing increases the output by about another factor of 40 per run to 96,000 data points. This estimation is based upon the number of distinct multiplexing (40) vectors reported by Church. The throughput estimation is nearly the million or so bases per run needed for efficient completion of the Human Genome Project in a reasonable time frame.
Other technologies which promise more resolution and speed such as ultra-thin gels and the use of longer glass plates with wedged spacers are physically limited in data compaction by geometry. Gel-filled capillaries presently offer an increase in resolution and dramatically decreases in running time but are also limited because of the technical complexities of creating large arrays and the difficulty in manufacturing and the limited lifetime of capillaries which can not be easily recast. Both these technologies employ on-the fly florescence detection through appropriate windows near the end of the electrophoresis gel. On-the-fly detection demands labeling of the ladder during the sequencing chemistry and are for all practical purposes incompatible with multiplexing where labeling occurs after electrophoresis utilizing specific labeled hybridization probes.
Multiplexing technology, at the present stage of development, is tedious and requires processing of individual nylon or nitrocellulose membranes upon which the multiplexed ladders are affixed. After standard slab gel sequencing the spatially resolved ladders need to be blotted out of the gel onto deposition membranes prior to the multiplexing development procedures involving labeled sequence specific DNA probes. Direct blotting runs the ladders completely off the electrophoresis gel onto a moving substrate of deposition membrane, and has the advantage of additional resolution since all the fragments must run the total length of the gel which affords more separation before being eluted onto the membrane. The downstream processing problems associated with handling and repeated washing and probings necessary for multiplexing are not, however, alleviated by direct blotting alone but are greatly reduced by using the drum device of the instant invention.